www.sciencemag.org/content/348/6233/444/suppl/DC1

Supplementary Material for

Translational tuning optimizes nascent protein folding in cells

Soo Jung Kim, Jae Seok Yoon, Hideki Shishido, Zhongying Yang, LeeAnn A. Rooney, Jose M. Barral, William R. Skach*

*Corresponding author. E-mail: skachw@ohsu.edu

Published 24 April 2015, Science 348, 444 (2015)

DOI: 10.1126/science.aaa3974

This PDF file includes:

Material and Methods Figs. S1 to S11 Table S1 and S2 Full Reference List

2

Materials and Methods Plasmids

CFP-NBD1 and non-fluorescent CFP fusion constructs for in vitro translation were generated as described previously (19). Amber stop codons (UAG) were engineered at indicated sites in NBD1 by PCR overlap extension and are described elsewhere (19, 20, 27). To enhance read-through efficiency at D567UAG and N597UAG, synonymous codon substitutions were introduced at Ala566 and Ala596 (–1 codon position) by changing GCU to GCA (33). The I601P mutation was introduced in pcDNA3-CFTR (34) and CFP-NBD1-D567TAG (19) by PCR overlap extension using complementary sense and antisense oligonucleotides: +CFTR I601P: 5’-AAC AAA ACT AGG CCA TTG GTC ACT TCT AAA ATG G-3’ and -CFTR I601P: 5’-AGT GAC CAA TGG CCT AGT TTT GTT AGC CAT C-3’. PCR fragment was subcloned into SphI and HpaI sites of pcDNA3-CFTR, and into SphI and SpeI sites of CFP-NBD1 D567TAG (19). Synonymous substitutions of CFTR codons 525 to 593 or 553 to 593 were introduced into pcDNA3-CFTR (34) by PCR overlap extension using synthetic oligonucleotides. Synonymous codon changes for residues 501-540 were introduced into pcDNA3-CFTR in a similar manner (34). The resulting ‘Fast’ and ‘Slow’ sequences are shown in Fig. S5. All PCR amplified and cloned regions of resulting plasmids were verified by DNA sequencing. In vitro transcription and translation

RNA transcripts were synthesized in vitro from PCR-amplified cDNA using SP6 RNA polymerase as described elsewhere (19, 20, 35). Briefly, transcription was carried out at 40°C for 2 h in a solution containing 80 mM HEPES-KOH/pH7.6; 16 mM MgCl2; 3 mM each of ATP, CTP, UTP, and GTP; 40 mM DTT; 2 mM spermidine; and 0.2 U/µl RNase inhibitor. RNA was precipitated with 4.5 M LiCl, 25 mM EDTA at -20°C and centrifuged at 16,000 x g at 4°C. The RNA pellet was resuspended in RNase free H2O. Purified RNA (60 ng/µl) was translated for 72 min at 24°C in reactions containing 40% rabbit reticulocyte lysate (RRL), 20 mM HEPES-KOH/pH7.6; 100 mM KOAc; 1.2 mM Mg(OAc)2; 50 µM each of 20 amino acids; 1 mM ATP; 1 mM GTP; 15 mM creatine phosphate; 2 mM DTT; 0.15 mM spermidine; 20 ng/µl bovine tRNA; 40 ng/µl creatine kinase; and 0.12 U/µl RNase inhibitor. For autoradiography, methionine was omitted, and Trans35S-label (MP Biomedicals) was added to translation reactions as described (27, 35). For FRET experiments, four parallel translation reactions were performed in the presence of synthetic amber suppressor tRNAs: [14C]Lys-tRNAamb or 7-nitrobenz-2-oxa-1,3-diazol-[14C]Lys-tRNAamb that was synthesized as described previously (19, 36). An RNA aptamer (37) that inhibits translation termination factor(s) eRF1/eRF3 was also added (1~2 µM) to improve stop codon read-through as described (see also Fig. S2). Translation reactions included: Donor only - translated in the presence of [14C]Lys-tRNAamb, Donor + Acceptor - translated in the presence of εNBD-[14C]Lys-tRNAamb, and two control reactions programmed with transcripts encoding a non-fluorescent version of ECFP (19) lacking a UAG codon. Ribosome nascent chain complexes (RNCs) from blank reactions were used to correct for background fluorescence and non-specific binding of [14C]Lys and εNBD-[14C]Lys as described (19).

3

Fluorescence measurements RNCs containing donor and acceptor probes were purified from 250 µl translation

reactions at 4°C by size exclusion chromatography using Sepharose CL-6B column equilibrated in buffer containing 100 mM KOAc, 10 mM MgCl2 and 40 mM HEPES-KOH/pH 7.5. CFP fluorescence emission spectra (λex = 430 nm, λem = 450 ~ 600 nm) were obtained at 4°C unless otherwise specified, using a Fluorolog 3-22 fluorometer (HORIBA Jobin Yvon, Edison, NJ). Nascent chains were released from the ribosome by addition of 200 µg/ml RNase A and fluorescence was measured at 24°C in column buffer plus 3 mM ATP. At the end of each experiment, the concentration of polypeptides in donor and donor + acceptor samples were determined by 14C-scintillation counting (ScintiSafe scintillation fluid, Fisher Scientific and LS6500 Beckman Scintillation counter) using the following equation: [RNC] = (cpmS-cpmB)/(CE x SA x vol) where [RNC] is the ribosome-nascent chain complexes concentration in nM. cpmS and cpmB are 14C counts/min from sample and blank reactions, respectively. CE is counting efficiency (95%). SA is the specific activity of [14C]Lys in dpm/pmol, and vol is the sample volume in ml. FRET efficiency was then calculated from the 14C-corrected acceptor-induced decrease in CFP fluorescence intensity (λex = 430 nm, λex = 475 nm) using the equation:

EFRET (%)= 1 – FDA/FD * 100 where FDA and FD are net 14C-corrected emission intensities per nM nascent chain in donor+acceptor and donor samples, respectively. Note that FA (Fluorescence of acceptor only) is undetectable under these conditions as shown previously (19) and hence omitted from our calculations. Note also that the refractive index, donor quantum yield, and spectral overlap of donor and acceptor fluorophores are constant in this system (18, 19) and that the potential uncertainty in R0 due to fluorophore dipole orientation in nascent chains as determined by fluorescence anisotropy is likely small (usually near ~10%) (19). Thus, while it is not possible to measure absolute distances using this approach due to the dynamic nature of the system, changes in FRET that occur as a result of the acceptor incorporation site, amino acid sequence, translation rate, nascent chain length, and presence of the ribosome, primarily result from changes in the linear distance separating donor and acceptor fluorophores. Translation elongation rate profiles

Relative translation elongation rates were calculated using the algorithm of Spencer et al. (10) that are based on fractional abundance of isoacceptor tRNAs and the nature of the codon-anticodon interaction, depending on whether each codon is translated by strict Watson-Crick base pairing, wobble base pairing, or both. Standard penalties for suboptimal energetics of non-Watson-Crick (wobble) codon-tRNA H-bonding interactions (Table S1) were derived from in vivo measurements (10, 37-39). Relative tRNA abundance was estimated by dividing gene copy number for each isoacceptor

4

(Genomic tRNA Database, http://gtrnadb.ucsc.edu (41)) by the number of tRNA genes within the individual synonymous codon group. Calculations for isoacceptor take into account that in eukaryotes, the A34 position of tRNAs is modified to inosine (I), which can base pair in a Watson-Crick-like fashion with cytosine, and in a non-Watson-Crick fashion with U and A. We therefore penalize non-Watson-Crick interactions (U:I and A:I) by a factor of 3 and leave C:I interactions unpenalized. As presented in Table S1, codons that end in U, e.g. Ala-5’GCU3’, which would normally be decoded by 5’AGC3’-bearing tRNAs, are instead decoded by 5’IGC3’-bearing tRNAs. Since the U:I is a non-Watson-Crick interaction, it is penalized, and thus the 0.674 value becomes 0.2248. Ala-5’GCU3’ can also be decoded by 5’GGC3’ via non-Watson-Crick interactions, but in humans there are none, and thus the value becomes 0.2248 + 0 = 0.2248. On the other hand, the Ala-5’GCC3’ codon could be decoded by 5’GGC3’-bearing tRNAs (none in humans = 0) and also by the modified 5’IGC3’-bearing tRNAs. Since the C:I interaction is Watson-Crick-like, we do not penalize it, and the value then becomes 0 + 0.6744 = 0.6744. We realize that this may appear paradoxical, but our calculations are designed to reflect the actual tRNA species available in cells that can pair with a particular codon. They take into account three key features, tRNAs present, the A34I modification, and W-C and non-W-C base pairing efficiency. While this approach could potentially be affected by differential tRNA gene expression, it is supported by a high positive correlation between gene copy and tRNA abundance (10, 40, 42). The relative elongation rate values (v, Table S1) for each of the 61 potential codons were then used to calculate the average translation elongation rate, plotted at the center residue of a 15 aa moving window of human CFTR NBD1 coding sequence. CFTR expression in mammalian cells

Human embryonic kidney (HEK) 293T cells were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Thermo Scientific) and penicillin/streptomycin (Invitrogen). 5 × 105 cells were seeded in six-well plates and transfected one day later with equal amounts (1-2.5 µg) of pcDNA3-NBD1 or pcDNA3-CFTR vector containing wild-type (WT) or synonymous ‘Fast’ and ‘Slow’ coding sequences as indicated. HEK 293T cells were co-transfected (Lipofectamine 2000, Invitrogen) with 50 ng of pEGFPN3 (Clontech) and blotted for GFP to confirm equivalent transfection efficiencies. Cells were analyzed 24 or 48 h after transfection for pulse-chase/short-pulse analyses or immunoblotting, respectively. Harvested cells were lysed for 20 min in 600 µl of ice-cold RIPA buffer (20 mM HEPES-NaOH/pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) containing complete protease inhibitor mixture (Roche Applied Science). Insoluble material was collected by centrifugation at 16,000 × g at 4°C for 15 min, rinsed once with Dulbecco's phosphate-buffered saline (Invitrogen) and solubilized at room temperature in 20 µl of 20 mM Tris-HCl/pH 7.4, 1% SDS for 20 min prior to adding 180 µl RIPA buffer. Alternatively, total cell lysate was collected by adding RIPA buffer

5

containing 1% SDS for 20 min on ice, and cell debris was removed by centrifugation at 16,000 x g, 4°C for 15 min. Immunoblotting; cell lysate (40 µg soluble protein and 8 µg insoluble protein) was separated by SDS–PAGE transferred to PVDF membrane (Millipore) and immunoblotted using the following primary antibodies: 1) mouse anti-CFTR antibody M3A7 (Millipore 1:5,000), 2) rabbit anti-β-actin (Cell Signaling 1:5,000), or 3) rabbit anti-GFP (Clontech, 1:5,000) and secondary antibodies: Alexa Fluor 680 goat anti-mouse IgG (Invitrogen 1:5,000) or Alexa Fluor 790 goat anti-rabbit IgG (Invitrogen 1:5,000). Blots were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed using accompanying image analysis software (version 3.0). Pulse-chase/short-pulse experiments; transfected cells were incubated for 30 min in cysteine and methionine-free medium, pulsed labeled with 30 µCi Trans35S-label (MP Biomedicals)/well, and chased for indicated times in fresh complete medium. To evaluate translation rates, cells were pulsed with 50 µCi Trans35S-label/well and harvested directly at the indicated short times without chase. Both RIPA soluble and insoluble fractions of pulse-chase samples were incubated with anti-CFTR antibody 3G11 (a generous gift of the CFTR Folding Consortium) overnight at 4°C, and then for 2 h at 4°C after addition of Affi-Gel Protein G (Bio-Rad). Protein G beads were washed five times with RIPA buffer and three times with Tris-buffered saline (20 mM Tris-HCl/pH 7.5, 137 mM NaCl) and mixed with SDS-PAGE sample buffer, followed by separation on SDS–PAGE. The radio labeled bands were imaged and analyzed with the Personal FX phosphor imager and Quantity One software (Bio-Rad). Real-time quantitative PCR

The mRNA levels of WT, Slow(525-593), and Fast(525-593) NBD1 were analyzed by the real-time quantitative PCR. Total RNA was extracted 24 h after transfection by Trizol (Invitrogen) and was treated by DNase I in order to remove DNA contamination according to manufacturer’s instructions. Equal amounts of the RNA samples (100 ng) were then reverse transcribed by using an iScript reverse transcriptase supermix (Bio-Rad), and the cDNA was amplified with specific primers, an iQ SYBR green supermix (Bio-Rad), and the Chrome4 real-time PCR detector (Bio-Rad). The primers used were as follows: NBD1, forward primer 5’-TCT GGG AGG AGG GAT TTG GG -3’; reverse primer 5’-GTG AAG TCT TGC CTG CTC CA -3’; and β–actin, forward primer 5’-GTC ACC AAC TGG GAC GAC AT-3’, reverse primer 5’-GAG GCG TAC AGG GAT AGC AC-3’. Slow(525-593) or Fast(525-593) NBD1 mRNA level, normalized to housekeeping gene β–actin and relative to the WT NBD1 sample, was calculated as 2-ΔΔCt , where ΔΔCt = (Ct Slow or Fast NBD1 - β–actin) - (Ct WT NBD1 - β–actin). Purification of WT and Fast(525-593) NBD1 and mass spectrometry protein sequencing

HEK 293T cells (7.5 × 106) were seeded in 150 mm dishes and transfected one day later with equal amounts (17 µg) of pcDNA3-NBD1 vector containing WT or synonymous Fast(525-593) coding sequences as indicated. Transfected cells were cultured for 24 h, harvested, and lysed for 20 min in 3 ml of ice-cold RIPA buffer containing complete protease inhibitor mixture (Roche Applied Science). After cell debris was

6

removed by centrifugation at 16,000 x g, 4°C for 15 min, total cell lysates (10 mg) were pre-cleared by incubation with Protein A beads (100 µl slurry) (Bio-Rad) for 2 h at 4°C. The pre-cleared cell lysates were incubated with anti-CFTR antibody 3G11 (10 µg) overnight at 4°C, and then for 4 h at 4°C after addition of Affi-Gel Protein G (100 µl slurry) (Bio-Rad). Protein G beads were washed five times with RIPA buffer and three times with Tris-buffered saline (20 mM Tris-HCl/pH 7.5, 137 mM NaCl) and mixed with SDS-PAGE sample buffer, followed by separation on SDS–PAGE. Excised NBD1 protein bands were reduced with DTT, alkylated with iodoacetamide, and digested overnight with trypsin, Asp-N, or Lys-C endoproteinase (43). Trypsin, or Asp-N digested peptides were separated using a NanoAcquity UPLC system, 75 µm x 25 cm BEH UPLC column containing 1.7 µm C18 resin (Waters), 300 nl/min flow rate, and 7.5 - 30% acetonitrile gradient over 60 min in a mobile phase containing 0.1% formic acid. Peptides were analyzed with a LTQ Velos dual pressure linear ion trap mass spectrometer (Thermo Scientific) using electrospray ionization and Captive Spray Source (Michrom Biosciences). For better identification of relatively large sized peptides, NBD1 digests by Lys-C were analyzed using a high resolution Orbitrap Fusion mass spectrometer, UltiMate 3000 nano UPLC system, 75 µm x 25 cm PepMap RSLC UPLC EasySpray column containing 2 µm C18 resin (Thermo Scientific), and chromatography conditions as described above. Protein Discoverer software (version 1.4, Thermo Scientific) was used to analyze the data from the mass spectrometers. Sequest HT was used to search LC-MS/MS spectra from NBD1 samples against a protein database containing human sequences from the Swiss-Prot database (downloaded May 2014) and 179 common contaminant sequences. The CFTR-NBD1 sequence (residues 389 to 673) was substituted for the full-length CFTR in the database to obtain accurate coverage data. Sequest HT searches for all samples were performed with trypsin, Asp-N, or Lys-C enzyme specificity. 2.0 Da for monoisotopic parent ion mass tolerance and 0.6 Da for monoisotopic fragment ion mass tolerance were used to match spectra generated by the LTQ mass spectrometer. Respectively, 10 ppm and 0.6 Da tolerances were used to match spectra generated by the Orbitrap mass spectrometer. A static modification of +57.021 Da was added to all cysteine residues and a variable modification of +15.995 to methionine. Search results were filtered to control peptide false discovery using Percolator (44) contained within Protein Discoverer 1.4 software and only peptides with q-values of 0.01 or less were accepted, corresponding to a false discovery rate of below 1%.

7

Acceptor-[14C]Lys

B (14C) B (Acceptor-14C)

D DA

NF-CFP NF-CFP

A

CFP CFP

[14C]Lys

Raw Emission Spectra

Rel

ativ

e Fl

uore

scen

ce

Inte

nsity

Net Emission Spectra

B

0.0

0.2

0.4

0.6

0.8

1.0

450 475 500 525 550 575 600

0.0

0.2

0.4

0.6

0.8

1.0

450 475 500 525 550 575 600

0.0

0.2

0.4

0.6

0.8

1.0

450 475 500 525 550 575 600

14C-Corrected Spectra

Rel

ativ

e Fl

uore

scen

ce

Inte

nsity

N

orm

aliz

ed

Fluo

resc

ence

/nM

Wavelength (nm)

D

DA

B

D

DA

D

DA

Fig. S1. Experimental FRET setup. (A) Schematic of RNCs generated by in vitro translation reactions. Blank translations (14C and Acceptor-14C) were programmed with transcript encoding non-fluorescent (NF) CFP-NBD1 fusion protein (described in (19)) lacking a UAG codon and translated in the presence of either [14C]Lys-tRNAamb or εNBD-[14C]Lys-tRNAamb, respectively. Donor only (D) and Donor+Acceptor (DA) samples were translated from transcripts containing a UAG codon in the presence of [14C]Lys-tRNAamb or εNBD-[14C]Lys-tRNAamb, respectively. (B) Typical CFP fluorescence spectra obtained from CFP-NBD1-D567UAG truncated at residue 744. Top panel: Raw emission spectra (λex = 430 nm, λem = 450-600 nm). Blank spectra are superimposed. Emission peak at 510 nm is from water Raman scatter. Middle panel shows net CFP emission spectra after background subtraction. Bottom panel shows CFP emission spectra (photons/sec/nM protein) corrected for nascent chain concentration in nM calculated from [14C]Lys content based on 14C-scintillation counting as described in Materials and Methods.

8

C

D/DA ratio

EFR

ET

(%)

40

30

20

10

0 0.7 0.8 0.9 1.1 1.2 1.3 1.0

D567UAG-744

*Asp567 D DA

46 KD -

66 KD -

30 KD -

B UAG site

46 KD -

66 KD -

30 KD -

Thr389 - D DA B

A Arg4

50

Arg487

Asp56

7

UAG -

46 KD

66 KD

30 KD

aptamer - + - + + UAG

46 KD

66 KD

30 KD

Arg 487

Asp 567

*Asp 567

Fig. S2. Effect of ribosome stalling on FRET. (A) Autoradiograms of CFP-NBD1 (truncation 744) containing UAG stop codons translated in vitro in the presence of εNBD-[14C]Lys-tRNAamb (left) and an eRF1/eRF3 inhibitor (aptamer, right) as indicated (37). Filled and open circles designate polypeptides that terminate at or read-through the UAG codon, respectively. Asterisk indicates synonymous codon change (GCU to GCA) at Ala566 to improve read-through efficiency (33). (B) Autoradiogram of [35S]Met-labeled translation products for CFP-NBD1 constructs containing indicated UAG stop codon. Polypeptides that terminate or read-through the UAG codon are indicated by filled and open circles, respectively. In our system, nascent chains that fail to incorporate the acceptor probe at the UAG codon are released from the ribosome and removed by size exclusion chromatography (19, 27). This allows us to isolate uniform intact RNCs that theoretically contain an equal (1:1) ratio of acceptor and donor probes. However, as the distance between the fusion site and UAG codon increases, ribosome stalls between CFP and the UAG codon generate a small

9

fraction of partial-length polypeptides that contain CFP but lack the acceptor probe (denoted by brackets). Lacking an acceptor probe, these RNCs exhibit a FRET value of zero. Due to the absence of a [14C]Lys residue, however, they reduce the calculated RNC concentration in the sample and proportionately raise the measured donor fluorescence intensity (pps/nM). In general, stalling has little effect other than a slight reduction in the measured FRET value. (C) However, if read-through efficiency at the UAG codon differs for [14C]Lys-tRNA (D) and εNBD-[14C]Lys-tRNA (DA) samples in an otherwise identical experiment, then stalled RNCs in D and DA samples affect FRET. High D/DA ratios (>1.2) underestimate FRET, whereas low D/DA ratios (<0.8) overestimate FRET. This is because the relative fraction of stalled polypeptides (with zero FRET) in the sample, is dependent on both the number of stalls and the number of polypeptides that actually read-through the stop codon. To compensate for this phenomenon, the translation termination factor eRF1/eRF3 was inhibited with an RNA aptamer (37) to equalize read-through efficiency of suppressor tRNAs, and only samples with D/DA ratio between 0.85 and 1.15 were used in this study.

10

B C A R450UAG E

FRE

T (%

) 50 40 30 20 10 0 -10 -5 0 5 10

release

-10 -5 0 5 10

506 500

Time (min)

EFR

ET

(%)

50 40 30 20 10 0 -10 -5 0 5 10

Time (min) -10 -5 0 5 10

515 535

R487UAG

EFR

ET

(%)

50 40 30 20 10 0 -10 -5 0 5 10

release

-10 -5 0 5 10

535 500

Time (min) Time (min)

EFR

ET

(%)

50 40 30 20 10 0 -10 -5 0 5 10 -10 -5 0 5 10

550 584

D567UAG 40

30

20

10

0

EFR

ET

(%)

release

-10 -5 0 5 10 -10 -5 0 5 10

614 584

Time (min)

40

30

20

10

0

EFR

ET

(%)

-10 -5 0 5 10 Time (min)

-10 -5 0 5 10

624 654

D

EFR

ET

(%)

50

40

30

20

10

0 552 548 550 552 548 550

bound

R487UAG

released

EFR

ET

(%)

40

30

20

10

0 616 612 614 616 612 614

D567UAG

Truncation site

Fig. S3. Differential effect of ribosome on NBD1 subdomain compaction. (A) EFRET obtained prior to and following ribosome release (T=0) of nascent CFP-NBD1-R450UAG polypeptides at indicated truncation (mean, N ≥ 2) compared to corresponding ribosome-bound polypeptides (dotted line from Fig. 1D). (B) EFRET obtained for CFP-NBD1-R487UAG polypeptides before and after ribosome release as in panel A (mean, N ≥ 3). (C) EFRET for CFP-NBD1-D567UAG obtained as in panels A and B (mean, N ≥ 3) (D) FRET efficiencies of ribosome bound and released CFP-NBD1-R487UAG (top) and D567UAG (bottom) polypeptide truncated at 2 amino acids increments (mean N ≥ 2).

11

A

40

30

20

10

0

EFR

ET(%

)

WT I601P WT I601P

D567UAG -744

D567UAG -654

GFP

B

CFTR

β-actin

WT I601P

─ C

─ B

250 KD-

130 KD-

36 KD- 28 KD-

Fig. S4. Effect of I601P in CFTR processing. (A) FRET efficiency of in vitro translated CFP-NBD1-D657UAG showing that proline substitution (I601P) into the S8 β-strand reduces efficiency of S7 positioning in truncated and full-length NBD1, residues 654 and 744, respectively (mean N ≥ 3, ± SEM). (B) Immunoblot showing that the I601P mutation also disrupts intracellular trafficking of full-length CFTR expressed in HEK 293T cells by eliminating production of the mature, complex glycosylated Band C.

12

Fig. S5. Synonymous codon substitutions in human CFTR NBD1. Human wild-type CFTR DNA sequence showing synonymous substitutions (highlighted in yellow) used to generate Slow/Fast(525-593) and Slow/Fast(501-540) constructs. Slow(525-593) substitutions were introduced at 40 codons whereas Fast(525-593) substitutions were introduced at 57 codons. Slow(501-540) or Fast(501-540) substitutions were introduced at 22 or 26 codons, respectively.

13

2.0�

1.5�

1.0�

0.5�

0�

Rel

ativ

e N

BD

1 m

RN

A

(Fol

d, 2

n )

WT�S(525-593) �F(525-593)�

Fig. S6. mRNA levels of WT, Slow(525-593), and Fast(525-593) NBD1 expressed in HEK 293T cells. The relative mRNA levels of WT, Slow(525-593), and Fast(525-593) NBD1 were analyzed 24 h after transfection by the real-time quantitative PCR. Graph shows relative amount of Slow(525-593) or Fast(525-593) NBD1 mRNA compared to WT (N = 3, ± SEM or average of N = 2).

14

A total cell lysate

input (0.1%)

NT �

WT �

Fast

(525

-593

)�

IP elute

36 KD-

28 KD-

17 KD-

55 KD-

Mar

ker �

NT �

WT �

Fast

(525

-593

)�

━ NBD1

B

━ 3G11 Ab L-chain

━ 3G11 Ab H-chain

━ #

15

Fig. S7. Mass spectrometry protein sequencing of WT and Fast(525-593) NBD1. (A) Silver stained gels of total cell lysates and immunoprecipitation (IP) elutes from non-transfected (NT), WT, and Fast(525-593) NBD1 transiently expressed in HEK 293T cells. # indicates non-specific protein. (B) Sequence coverage of NBD1 (yellow). Individual peptides identified after trypsin (red), Lys-C (blue), or Asp-N (black) digestions are underlined. Synonymous codon substitutions from residue 525 to 593 indicated by bold letters were completely identified in WT and Fast(525-593) NBD1.

16

100�80�60�40�20�

0�Sol

uble

(%)

0� 1� 2� 4�

15�

10�

5�

0�Inso

lubl

e (%

) 0� 1� 2� 3� 4�

soluble insoluble 0� 1� 2� 4�

WT

Slow (525-593)

Chase (h)

Time (h) Time (h)

Fast (525-593)

WT�Slow�Fast�

0� 1� 2� 3� 4�

- NBD1

- NBD1

- NBD1

28KD-

28KD-

28KD-

Fig. S8. Comparison of WT, Slow(525-593), and Fast(525-593) NBD1 synthesis rate in HEK 293T cells. [35S]Met-labeled NBD1 immunoprecipitated from RIPA soluble and insoluble fractions. Graphs show percentage of NBD1 recovered relative to T=0 (N ≥ 3 ± SEM).

17

CFTR

β-actin

GFP

Rel

ativ

e C

FTR

(Fol

d)

soluble insoluble 8�

6�

4�

2�

0�

ΔF5

08�

ΔF/

S(5

25-5

93)�

ΔF/

F (52

5-59

3)�

ΔF5

08�

ΔF/

S(5

25-5

93)�

ΔF/

F (52

5-59

3)�

ΔF5

08�

ΔF/

S(5

25-5

93)�

ΔF/

F (52

5-59

3)�

━ B

soluble insoluble total A

B ΔF/S

(525-593)�ΔF/F

(525-593) �

ΔF�

ΔF/S(525-593)�

ΔF/F(525-593) �

ΔF�

250 KD-

130 KD-

36 KD-

28 KD-

Fig. S9. Effect of Fast and Slow synonymous codon substitutions on ∆F508 CFTR processing. (A) Immunoblot of ∆F508 (∆F), ∆F508-Slow(525-593) (∆F/S(525-593)), and ∆F508-Fast(525-593) (∆F/F(525-593)) CFTR transiently expressed in HEK 293T cells. Cells were lysed in SDS or RIPA buffer to recover total and soluble protein fractions, respectively. Insoluble fraction was obtained by SDS-solubilization of the RIPA-insoluble pellet. GFP and β-actin blots show transfection and loading controls, respectively. (B) Graph shows relative amount of indicated proteins recovered in soluble and insoluble fraction for indicated constructs (N ≥ 4 ± SEM).

18

Fig. S10. Fast(501-540) synonymous codon substitutions do not result in CFTR aggregation. (A) Predicted translation elongation rate (10) calculated as 15 aa moving window average, for WT NBD1 (black), Slow(501-540) (red), and Fast(501-540) (blue), aligned with secondary structural elements as they emerge from the ribosome. (B) Immunoblot of soluble and insoluble WT, Slow(501-540), and Fast(501-540) CFTR. Graph shows quantitation (N = 5 ± SEM) as in Fig. 3C.

19

A

B

Truncation site

450 550 650 750 500 600 700

EFR

ET

(%)

50

40

30

20

10

0

WT R487UAG

ΔF508 R487UAG

WT D567UAG

Truncation site

450 550 650 750 500 600 700

EFR

ET

(%)

40

30

20

10

0

ΔF508 D567UAG

Truncation site

450 550 650 750 500 600 700

EFR

ET

(%)

50

40

30

20

10

0

WT R487UAG

D529F R487UAG

C

Fig. S11. Effect of D529F and ΔF508 on FRET. FRET profiles of D529F R487UAG (A), ∆F508 R487UAG (B) ∆F508 D567UAG (C) together with WT NBD1. WT data from Fig. 1D are plotted here for direct comparison.

20

Table S1. Predicted relative translation elongation rates for human codons

AminoAcid

mRNA codon

(5' to 3')

Cognate tRNAs Isoacceptor gene fraction

Cognate tRNA contribution

(A)

Other compatible tRNAs Other compatible

tRNA contribution (B)

Relative translation elongation rate (v)

(=A+B) anticodon (5' to 3')

gene copy #

anticodon (5' to 3')

gene copy #

Ala

GCU AGC 29 0.6744 *0.2248 0.2248 GCC GGC 0 0.0000 AGC 29 0.6744 0.6744 GCA UGC 9 0.2093 0.2093 AGC 29 *0.2248 0.4341 GCG CGC 5 0.1163 0.1163 UGC 9 *0.0698 0.1861

Gly

GGU ACC 0 0.0000 GCC 15 *0.1613 0.1613 GGC GCC 15 0.4839 0.4839 0.4839 GGA UCC 9 0.2903 0.2903 0.2903 GGG CCC 7 0.2258 0.2258 UCC 9 *0.0968 0.3226

Pro

CCU AGG 10 0.4762 *0.1587 0.1587 CCC GGG 0 0.0000 AGG 10 0.4762 0.4762 CCA UGG 7 0.3333 0.3333 AGG 10 *0.1587 0.4920 CCG CGG 4 0.1905 0.1905 UGG 7 *0.1111 0.3016

Thr

ACU AGU 10 0.4545 *0.1515 0.1515 ACC GGU 0 0.0000 AGU 10 0.4545 0.4545 ACA UGU 6 0.2727 0.2727 AGU 10 *0.1515 0.4242 ACG CGU 6 0.2727 0.2727 UGU 6 *0.0909 0.3636

Val

GUU AAC 11 0.3438 *0.1146 0.1146 GUC GAC 0 0.0000 AAC 11 0.3438 0.3438 GUA UAC 5 0.1563 0.1563 AAC 11 *0.1146 0.2709 GUG CAC 16 0.5000 0.5000 UAC 5 *0.0521 0.5521

Ser

UCU AGA 11 0.3929 *0.1310 0.1310 UCC GGA 0 0.0000 AGA 11 0.3929 0.3929 UCA UGA 5 0.1786 0.1786 AGA 11 *0.1310 0.3096 UCG CGA 4 0.1429 0.1429 UGA 5 *0.0595 0.2024 AGU ACU 0 0.0000 GCU 8 *0.0952 0.0952 AGC GCU 8 0.2857 0.2857 0.2857

Arg

CGU ACG 7 0.2500 *0.0833 0.0833 CGC GCG 0 0.0000 ACG 7 0.2500 0.2500 CGA UCG 6 0.2143 0.2143 ACG 7 *0.0833 0.2976 CGG CCG 4 0.1429 0.1429 UCG 6 *0.0714 0.2143 AGA UCU 6 0.2143 0.2143 0.2143 AGG CCU 5 0.1786 0.1786 UCU 6 *0.0714 0.2500

Leu

CUU AAG 12 0.3077 *0.1026 0.1026 CUC GAG 0 0.0000 AAG 12 0.3077 0.3077 CUA UAG 3 0.0769 0.0769 AAG 12 *0.1026 0.1795 CUG CAG 10 0.2564 0.2564 UAG 3 *0.0256 0.2820 UUA UAA 7 0.1795 0.1795 0.1795 UUG CAA 7 0.1795 0.1795 UAA 7 *0.0598 0.2393

Phe UUU AAA 0 0.0000 GAA 12 *0.3333 0.3333 UUC GAA 12 1.0000 1.0000 1.0000

Asn AAU AUU 2 0.0588 0.0588 GUU 32 *0.3137 0.3725 AAC GUU 32 0.9412 0.9412 AUU 2 0.0588 1.0000

Lys AAA UUU 16 0.4848 0.4848 0.4848 AAG CUU 17 0.5152 0.5152 UUU 16 *0.1616 0.6768

Asp GAU AUC 0 0.0000 GUC 19 *0.3333 0.3333 GAC GUC 19 1.0000 1.0000 1.0000

Glu GAA UUC 13 0.5000 0.5000 0.5000 GAG CUC 13 0.5000 0.5000 UUC 13 *0.1667 0.6667

His CAU AUG 0 0.0000 GUG 11 *0.3333 0.3333 CAC GUG 11 1.0000 1.0000 1.0000

Gln CAA UUG 11 0.3548 0.3548 0.3548 CAG CUG 20 0.6452 0.6452 UUG 11 *0.1183 0.7635

Ile AUU AAU 14 0.6364 *0.2121 GAU 3 *0.0455 0.2576 AUC GAU 3 0.1364 0.1364 AAU 14 0.6364 0.7728 AUA UAU 5 0.2273 0.2273 AAU 14 *0.2121 0.4394

Met AUG CAU 20 1.0000 1.0000 1.0000

Tyr UAU AUA 1 0.0667 0.0667 GUA 14 *0.3111 0.3778 UAC GUA 14 0.9333 0.9333 AUA 1 0.0667 1.0000

Cys UGU ACA 0 0.0000 GCA 30 *0.3333 0.3333 UGC GCA 30 1.0000 1.0000 1.0000

Trp UGG CCA 9 1.0000 1.0000 1.0000 *: Non-Watson-Crick interactions are penalized by a factor of 3.

21

Column labeled “Cognate tRNAs” shows the number of indicated tRNA genes in human genome (from http://gtrnadb.ucsc.edu (41)). “Isoacceptor gene fraction” is calculated as fraction of cognate tRNA genes relative to the total number of tRNA genes encoding for that amino acid. “Other compatible tRNAs” shows number of additionally indicated tRNA genes in the genome that are capable of decoding that codon. Columns labeled “Cognate tRNA contribution (A)”, and “Other compatible tRNA contribution (B)” show relative contribution of each tRNA pool towards translation rate of the indicated codon calculated from the gene fraction using either Watson-Crick base pairing or wobble codon base pairing (asterisk designates wobble base pairing interactions). Note that calculations take into account adenosine-to-inosine conversion at position 34 on the tRNA. “Relative translation elongation rate (v)” shows the sum contribution of cognate and additional tRNAs that are capable of decoding the indicated codon (v = A + B) according to Spencer et al. (10).

22

Table S2. Protein sequencing summary – identified peptides NBD1 Enzyme

eee Start Residue Sequence #PSMs q-Value

WT Trypsin 420 KTSNGDDSLFFSNFSLLGTPVLK 1 0 421 TSNGDDSLFFSNFSLLGTPVLK 3 0 443 DINFKIER 6 0.009 448 IERGQLLAVAGSTGAGK 4 0 451 GQLLAVAGSTGAGK 61 0 465 TSLLmmImGELEPSEGKIK 1 0 465 TSLLMmImGELEPSEGKIK 2 0.001 504 ENIIFGVSYDEYR 6 0 523 AcQLEEDISK 28 0 523 AcQLEEDISKFAEK 3 0 523 AcQLEEDISKFAEKDNIVLGEGGITLSGG

QR 1 0

533 FAEKDNIVLGEGGITLSGGQR 13 0 537 DNIVLGEGGITLSGGQR 9 0 561 AVYKDADLYLLDSPFGYLDVLTEK 2 0 565 DADLYLLDSPFGYLDVLTEK 1 0.001 585 EIFEScVcK 12 0.001 601 ILVTSKmEHLK 2 0 616 ILILHEGSSYFYGTFSELQNLQPDFSSK 1 0 644 LmGcDSFDQFSAER 14 0 644 LMGcDSFDQFSAER 4 0 644 LmGcDSFDQFSAERR 6 0.001 658 RNSILTETLHR 7 0 659 NSILTETLHR 12 0

Lys-C 421 TSNGDDSLFFSNFSLLGTPVLK 6 0 443 DINFKIERGQLLAVAGSTGAGK 2 0.003 448 IERGQLLAVAGSTGAGK 18 0 465 TSLLmmImGELEPSEGK 8 0 465 TSLLMmImGELEPSEGK 8 0 465 TSLLMmIMGELEPSEGK 2 0.004 482 IKHSGRISFcSQFSWImPGTIK 3 0 482 IKHSGRISFcSQFSWIMPGTIK 2 0 484 HSGRISFcSQFSWImPGTIK 6 0 484 HSGRISFcSQFSWIMPGTIK 4 0 504 ENIIFGVSYDEYRYRSVIK 22 0 523 AcQLEEDISK 14 0 537 DNIVLGEGGITLSGGQRARISLARAVYK 22 0 565 DADLYLLDSPFGYLDVLTEK 6 0 585 EIFEScVcK 14 0 599 TRILVTSK 5 0 616 ILILHEGSSYFYGTFSELQNLQPDFSSK 4 0

Asp-N 425 DDSLFFSNFSLLGTPVLK 1 0 513 DEYRYRSVIKAcQLEE 2 0.006 529 DISKFAEK 3 0.007 639 DFSSKLmGc 9 0 639 DFSSKLMGc 3 0.005 639 DFSSKLmGcDSF 2 0

c = carbamidomethly cysteine / m = oxidized methionine

(continued)

23

NBD1 Enzyme

eee Start Residue

## # #### ### # Sequence #PSMs q-Value

Fast (525-593)

Trypsin 420 KTSNGDDSLFFSNFSLLGTPVLK 2 0 421 TSNGDDSLFFSNFSLLGTPVLK 28 0 421 TSNGDDSLFFSNFSLLGTPVLKDINFK 1 0 443 DINFKIER 30 0.004 448 IERGQLLAVAGSTGAGK 10 0 451 GQLLAVAGSTGAGK 93 0 465 TSLLMMIMGELEPSEGK 2 0 465 TSLLmmImGELEPSEGK 7 0 465 TSLLMmImGELEPSEGK 3 0 465 TSLLMMImGELEPSEGK 1 0.002 465 TSLLMMIMGELEPSEGKIK 2 0 465 TSLLMmIMGELEPSEGKIK 2 0 465 TSLLmmImGELEPSEGKIK 3 0 465 TSLLMmImGELEPSEGKIK 3 0 488 ISFcSQFSWImPGTIK 5 0 488 ISFcSQFSWIMPGTIK 1 0 504 ENIIFGVSYDEYR 62 0 504 ENIIFGVSYDEYRYR 2 0 519 SVIKAcQLEEDISK 2 0 523 AcQLEEDISK 27 0 523 AcQLEEDISKFAEK 7 0 523 AcQLEEDISKFAEKDNIVLGEGGITLSGGQ

R 3 0

533 FAEKDNIVLGEGGITLSGGQR 66 0 537 DNIVLGEGGITLSGGQR 50 0 565 DADLYLLDSPFGYLDVLTEK 2 0 565 DADLYLLDSPFGYLDVLTEKEIFEScVcK 2 0.005 585 EIFEScVcK 14 0 612 KADKILILHEGSSYFYGTFSELQNLQPDFSSK 1 0.001 613 ADKILILHEGSSYFYGTFSELQNLQPDFSSK 1 0 616 ILILHEGSSYFYGTFSELQNLQPDFSSK 11 0 644 LmGcDSFDQFSAER 34 0 644 LMGcDSFDQFSAER 22 0 644 LMGcDSFDQFSAERR 13 0 644 LmGcDSFDQFSAERR 14 0 658 RNSILTETLHR 31 0 659 NSILTETLHR 70 0

Lys-C 421 TSNGDDSLFFSNFSLLGTPVLK 4 0 443 DINFKIERGQLLAVAGSTGAGK 2 0 448 IERGQLLAVAGSTGAGK 16 0 465 TSLLmmImGELEPSEGK 7 0 465 TSLLMmImGELEPSEGK 5 0 465 TSLLMMIMGELEPSEGK 1 0 482 IKHSGRISFcSQFSWImPGTIK 2 0 484 HSGRISFcSQFSWIMPGTIK 3 0 484 HSGRISFcSQFSWImPGTIK 7 0 504 ENIIFGVSYDEYRYRSVIK 16 0 523 AcQLEEDISK 9 0 537 DNIVLGEGGITLSGGQRARISLARAVYK 14 0 565 DADLYLLDSPFGYLDVLTEK 3 0 585 EIFEScVcK 12 0 599 TRILVTSK 3 0 616 ILILHEGSSYFYGTFSELQNLQPDFSSK 4 0

Asp-N 425 DDSLFFSNFSLLGTPVLK 1 0 426 DSLFFSNFSLLGTPVLK 2 0 513 DEYRYRSVIKAcQLEE 6 0.004 529 DISKFAEK 8 0 572 DSPFGYL 2 0.006 639 DFSSKLmGc 9 0 639 DFSSKLMGc 7 0 639 DFSSKLmGcDSF 3 0 639 DFSSKLMGcDSF 3 0

c = carbamidomethly cysteine / m = oxidized methionine

24

Unique NBD1 peptide sequences identified by LC-MS/MS analyses are listed. Enzymes used to generate NBD1 peptides are shown in column 2, along with initial residue number of the peptide (column 3). Amino acid sequence, number of peptide-spectrum matches (PSM), and q-value are indicated next to each unique peptide. The region encoding ‘Fast’ synonymous codon substitutions (residues 525-593) is indicated by bold letters. Matching peptides fully covering the synonymous codon region were identified in both WT and Fast(525-593) NBD1.

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